Hyperpolarized 13C NMR observation of lactate kinetics in skeletal muscle.
journal of experimental biology
2015; 218: 3308-3318
Metabolite-selective hyperpolarized (13)C imaging using extended chemical shift displacement at 9.4T.
Magnetic resonance imaging
The production of glycolytic end products, such as lactate, usually evokes the concept of a cellular shift from aerobic to anaerobic ATP generation and O2 insufficiency. In the classical view, muscle lactate must export to liver for clearance. Studies, however, indicate that lactate also forms under well-oxygenated conditions and have led investigators to postulate lactate shuttling from non-oxidative to oxidative muscle fiber, where it can serve as a precursor. Indeed, the intracellular lactate shuttle and the glycogen shunt hypotheses expand the vision to include a dynamic mobilization and utilization of lactate during a muscle contraction cycle. Testing the tenability of these provocative ideas during a rapid contraction cycle has posed a technical challenge. The present study herein reports the use of hyperpolarized [1-(13)C]lactate and [2-(13)C]pyruvate in dynamic nuclear polarization (DNP) NMR experiments to measure the rapid pyruvate and lactate kinetics in rat muscle. With a 2-s temporal resolution, (13)C DNP NMR detects both [1-(13)C]lactate and [2-(13)C]pyruvate kinetics in muscle. Infusing dichloroacetate to stimulate pyruvate dehydrogenase activity and shifts the kinetics toward oxidative metabolism. Bicarbonate formation increases sharply from [1-(13)C]lactate. Acetyl-L-carnitine, acetoacetate, and glutamate levels rise. Such a quick mobilization of pyruvate and lactate toward oxidative metabolism supports the postulated role of lactate in the glycogen shunt and the intracellular lactate shuttle models. The study introduces then an innovative DNP approach to measure metabolite transients, which will help delineate the cellular and physiological role of lactate and glycolytic end products.
View details for DOI 10.1242/jeb.123141
View details for PubMedID 26347554
Volumetric spiral chemical shift imaging of hyperpolarized [2-(13) c]pyruvate in a rat c6 glioma model.
Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine
To develop a technique for frequency-selective hyperpolarized (13)C metabolic imaging in ultra-high field strength which exploits the broad spatial chemical shift displacement in providing spectral and spatial selectivity.The spatial chemical shift displacement caused by the slice-selection gradient was utilized in acquiring metabolite-selective images. Interleaved images of different metabolites were acquired by reversing the polarity of the slice-selection gradient at every repetition time, while using a low-bandwidth radio-frequency excitation pulse to alternatingly shift the displaced excitation bands outside the imaging subject. Demonstration of this technique is presented using (1)H phantom and in vivo mouse renal hyperpolarized (13)C imaging experiments with conventional chemical shift imaging and fast low-angle shot sequences.From phantom and in vivo mouse studies, the spectral selectivity of the proposed method is readily demonstrated using results of chemical shift spectroscopic imaging, which displayed clearly delineated images of different metabolites. Imaging results using the proposed method without spectral encoding also showed effective separation while also providing high spatial resolution.This method provides a way to acquire spectrally selective hyperpolarized (13)C metabolic images in a simple implementation, and with potential ability to support combination with more elaborate readout methods for faster imaging.
View details for DOI 10.1016/j.mri.2015.12.023
View details for PubMedID 26707851
Assessing inflammatory liver injury in an acute CCl4 model using dynamic 3D metabolic imaging of hyperpolarized [1-(13) C]pyruvate.
NMR in biomedicine
MRS of hyperpolarized [2-(13) C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the (13) C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-(13) C]pyruvate in glioma-bearing brain.Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-(13) C]pyruvate, [2-(13) C]lactate, and [5-(13) C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-(13) C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-(13) C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity.The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-(13) C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain.Robust volumetric imaging with hyperpolarized [2-(13) C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate. Magn Reson Med, 2015. © 2015 Wiley Periodicals, Inc.
View details for DOI 10.1002/mrm.25766
View details for PubMedID 25946547
The feasibility of assessing branched-chain amino acid metabolism in cellular models of prostate cancer with hyperpolarized [1-C-13]-ketoisocaproate
MAGNETIC RESONANCE IMAGING
2014; 32 (7): 791-795
To facilitate diagnosis and staging of liver disease, sensitive and non-invasive methods for the measurement of liver metabolism are needed. This study used hyperpolarized (13) C-pyruvate to assess metabolic parameters in a CCl4 model of liver damage in rats. Dynamic 3D (13) C chemical shift imaging data from a volume covering kidney and liver were acquired from 8 control and 10 CCl4 -treated rats. At 12 time points at 5 s temporal resolution, we quantified the signal intensities and established time courses for pyruvate, alanine, and lactate. These measurements were compared with standard liver histology and an alanine transaminase (ALT) enzyme assay using liver tissue from the same animals. All CCl4 -treated but none of the control animals showed histological liver damage and elevated ALT enzyme levels. In agreement with these results, metabolic imaging revealed an increased alanine/pyruvate ratio in liver of CCl4 -treated rats, which is indicative of elevated ALT activity. Similarly, lactate/pyruvate ratios were higher in CCl4 -treated compared with control animals, demonstrating the presence of inflammation. No significant differences in metabolite ratios were observed in kidney or vasculature. Thus this work shows that metabolic imaging using (13) C-pyruvate can be a successful tool to non-invasively assess liver damage in vivo. Copyright © 2015 John Wiley & Sons, Ltd.
View details for DOI 10.1002/nbm.3431
View details for PubMedID 26474216
Dynamic Metabolic Imaging of Hyperpolarized [2-C-13]Pyruvate Using Spiral Chemical Shift Imaging with Alternating Spectral Band Excitation
MAGNETIC RESONANCE IN MEDICINE
2014; 71 (6): 2051-2058
Recent advancements in the field of hyperpolarized (13)C magnetic resonance spectroscopy (MRS) have yielded powerful techniques capable of real-time analysis of metabolic pathways. These non-invasive methods have increasingly shown application in impacting disease diagnosis and have further been employed in mechanistic studies of disease onset and progression. Our goals were to investigate branched-chain aminotransferase (BCAT) activity in prostate cancer with a novel molecular probe, hyperpolarized [1-(13)C]-2-ketoisocaproate ([1-(13)C]-KIC), and explore the potential of branched-chain amino acid (BCAA) metabolism to serve as a biomarker. Using traditional spectrophotometric assays, BCAT enzymatic activities were determined in vitro for various sources of prostate cancer (human, transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse and human cell lines). These preliminary studies indicated that low levels of BCAT activity were present in all models of prostate cancer but enzymatic levels are altered significantly in prostate cancer relative to healthy tissue. The MR spectroscopic studies were conducted with two cellular models (PC-3 and DU-145) that exhibited levels of BCAA metabolism comparable to the human disease state. Hyperpolarized [1-(13)C]-KIC was administered to prostate cancer cell lines, and the conversion of [1-(13)C]-KIC to the metabolic product, [1-(13)C]-leucine ([1-(13)C]-Leu), could be monitored via hyperpolarized (13)C MRS.
View details for DOI 10.1016/j.mri.2014.04.015
View details for Web of Science ID 000339531900001
View details for PubMedID 24907854
Hyperpolarized [1,4-C-13]-diethylsuccinate: a potential DNP substrate for in vivo metabolic imaging
NMR IN BIOMEDICINE
2014; 27 (3): 356-362
In contrast to [1-(13) C]pyruvate, hyperpolarized [2-(13) C]pyruvate permits the ability to follow the (13) C label beyond flux through pyruvate dehydrogenase complex and investigate the incorporation of acetyl-coenzyme A into different metabolic pathways. However, chemical shift imaging (CSI) with [2-(13) C]pyruvate is challenging owing to the large spectral dispersion of the resonances, which also leads to severe chemical shift displacement artifacts for slice-selective acquisitions.This study introduces a sequence for three-dimensional CSI of [2-(13) C]pyruvate using spectrally selective excitation of limited frequency bands containing a subset of metabolites. Dynamic CSI data were acquired alternately from multiple frequency bands in phantoms for sequence testing and in vivo in rat heart.Phantom experiments verified the radiofrequency pulse design and demonstrated that the signal behavior of each group of resonances was unaffected by excitation of the other frequency bands. Dynamic three-dimensional (13) C CSI data demonstrated the sequence capability to image pyruvate, lactate, acetylcarnitine, glutamate, and acetoacetate, enabling the analysis of organ-specific spectra and metabolite time courses.The presented method allows CSI of widely separated resonances without chemical shift displacement artifact, acquiring multiple frequency bands alternately to obtain dynamic time-course information. This approach enables robust imaging of downstream metabolic products of acetyl-coenzyme A with hyperpolarized [2-(13) C]pyruvate. Magn Reson Med, 2013. © 2013 Wiley Periodicals, Inc.
View details for DOI 10.1002/mrm.24871
View details for Web of Science ID 000336260900013
View details for PubMedID 23878057
In vivo investigation of cardiac metabolism in the rat using MRS of hyperpolarized [1-C-13] and [2-C-13]pyruvate
NMR IN BIOMEDICINE
2013; 26 (12): 1680-1687
The tricarboxylic acid (TCA) cycle performs an essential role in the regulation of energy and metabolism, and deficiencies in this pathway are commonly correlated with various diseases. However, the development of non-invasive techniques for the assessment of the cycle in vivo has remained challenging. In this work, the applicability of a novel imaging agent, [1,4-(13) C]-diethylsuccinate, for hyperpolarized (13) C metabolic imaging of the TCA cycle was explored. In vivo spectroscopic studies were conducted in conjunction with in vitro analyses to determine the metabolic fate of the imaging agent. Contrary to previous reports (Zacharias NM et al. J. Am. Chem. Soc. 2012; 134: 934-943), [(13) C]-labeled diethylsuccinate was primarily metabolized to succinate-derived products not originating from TCA cycle metabolism. These results illustrate potential issues of utilizing dialkyl ester analogs of TCA cycle intermediates as molecular probes for hyperpolarized (13) C metabolic imaging. Copyright © 2014 John Wiley & Sons, Ltd.
View details for DOI 10.1002/nbm.3071
View details for Web of Science ID 000330798100013
View details for PubMedID 24421249
Measuring mitochondrial metabolism in rat brain in vivo using MR Spectroscopy of hyperpolarized [2-C-13]pyruvate
NMR IN BIOMEDICINE
2013; 26 (10): 1197-1203
Hyperpolarized (13) C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl-coenzyme A (acetyl-CoA). [1-(13) C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in (13) C-bicarbonate production after dichloroacetate (DCA) administration. With [1-(13) C]pyruvate, the (13) C label is released as (13) CO2 /(13) C-bicarbonate, and, hence, does not allow us to follow the fate of acetyl-CoA. Pyruvate labeled in the C2 position has been used to track the (13) C label into the TCA (tricarboxylic acid) cycle and measure [5-(13) C]glutamate as well as study changes in [1-(13) C]acetylcarnitine with DCA and dobutamine. This work investigates changes in the metabolic fate of acetyl-CoA in response to metabolic interventions of DCA-induced increased PDC flux in the fed and fasted state, and increased cardiac workload with dobutamine in vivo in rat heart at two different pyruvate doses. DCA led to a modest increase in the (13) C labeling of [5-(13) C]glutamate, and a considerable increase in [1-(13) C]acetylcarnitine and [1,3-(13) C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2-(13) C]lactate, [2-(13) C]alanine and [5-(13) C]glutamate. The change in glutamate with dobutamine was observed using a high pyruvate dose but not with a low dose. The relative changes in the different metabolic products provide information about the relationship between PDC-mediated oxidation of pyruvate and its subsequent incorporation into the TCA cycle compared with other metabolic pathways. Using a high dose of pyruvate may provide an improved ability to observe changes in glutamate. Copyright © 2013 John Wiley & Sons, Ltd.
View details for DOI 10.1002/nbm.3003
View details for Web of Science ID 000327157400007
Effects of Isoflurane Anesthesia on Hyperpolarized C-13 Metabolic Measurements in Rat Brain
MAGNETIC RESONANCE IN MEDICINE
2013; 70 (4): 1117-1124
Metabolic response of glioma to dichloroacetate measured in vivo by hyperpolarized C-13 magnetic resonance spectroscopic imaging
2013; 15 (4): 433-441
Hyperpolarized [1-(13) C]pyruvate ([1-(13) C]Pyr) has been used to assess metabolism in healthy and diseased states, focusing on the downstream labeling of lactate (Lac), bicarbonate and alanine. Although hyperpolarized [2-(13) C]Pyr, which retains the labeled carbon when Pyr is converted to acetyl-coenzyme A, has been used successfully to assess mitochondrial metabolism in the heart, the application of [2-(13) C]Pyr in the study of brain metabolism has been limited to date, with Lac being the only downstream metabolic product reported previously. In this study, single-time-point chemical shift imaging data were acquired from rat brain in vivo. [5-(13) C]Glutamate, [1-(13) C]acetylcarnitine and [1-(13) C]citrate were detected in addition to resonances from [2-(13) C]Pyr and [2-(13) C]Lac. Brain metabolism was further investigated by infusing dichloroacetate, which upregulates Pyr flux to acetyl-coenzyme A. After dichloroacetate administration, a 40% increase in [5-(13) C]glutamate from 0.014 ± 0.004 to 0.020 ± 0.006 (p = 0.02), primarily from brain, and a trend to higher citrate (0.002 ± 0.001 to 0.004 ± 0.002) were detected, whereas [1-(13) C]acetylcarnitine was increased in peripheral tissues. This study demonstrates, for the first time, that hyperpolarized [2-(13) C]Pyr can be used for the in vivo investigation of mitochondrial function and tricarboxylic acid cycle metabolism in brain. Copyright © 2013 John Wiley & Sons, Ltd.
View details for DOI 10.1002/nbm.2935
View details for Web of Science ID 000324462100001
Metabolite kinetics in C6 rat glioma model using magnetic resonance spectroscopic imaging of hyperpolarized [1-13C]pyruvate
MAGNETIC RESONANCE IN MEDICINE
2012; 68 (6): 1886-1893
The metabolic phenotype that derives disproportionate energy via glycolysis in solid tumors, including glioma, leads to elevated lactate labeling in metabolic imaging using hyperpolarized [1-(13)C]pyruvate. Although the pyruvate dehydrogenase (PDH)-mediated flux from pyruvate to acetyl coenzyme A can be indirectly measured through the detection of carbon-13 ((13)C)-labeled bicarbonate, it has proven difficult to visualize (13)C-bicarbonate at high enough levels from injected [1-(13)C]pyruvate for quantitative analysis in brain. The aim of this study is to improve the detection of (13)C-labeled metabolites, in particular bicarbonate, in glioma and normal brain in vivo and to measure the metabolic response to dichloroacetate, which upregulates PDH activity.An optimized protocol for chemical shift imaging and high concentration of hyperpolarized [1-(13)C]pyruvate were used to improve measurements of lactate and bicarbonate in C6 glioma-transplanted rat brains. Hyperpolarized [1-(13)C]pyruvate was injected before and 45 min after dichloroacetate infusion. Metabolite ratios of lactate to bicarbonate were calculated to provide improved metrics for characterizing tumor metabolism.Glioma and normal brain were well differentiated by lactate-to-bicarbonate ratio (P = .002, n = 5) as well as bicarbonate (P = .0002) and lactate (P = .001), and a stronger response to dichloroacetate was observed in glioma than in normal brain.Our results clearly demonstrate for the first time the feasibility of quantitatively detecting (13)C-bicarbonate in tumor-bearing rat brain in vivo, permitting the measurement of dichloroacetate-modulated changes in PDH flux. The simultaneous detection of lactate and bicarbonate provides a tool for a more comprehensive analysis of glioma metabolism and the assessment of metabolic agents as anti-brain cancer drugs.
View details for DOI 10.1093/neuonc/nos319
View details for Web of Science ID 000316965600005
View details for PubMedID 23328814
Application of hyperpolarized [1-13C]lactate for the in vivo investigation of cardiac metabolism
NMR IN BIOMEDICINE
2012; 25 (10): 1119-1124
In addition to an increased lactate-to-pyruvate ratio, altered metabolism of a malignant glioma can be further characterized by its kinetics. Spatially resolved dynamic data of pyruvate and lactate from C6-implanted female Sprague-Dawley rat brain were acquired using a spiral chemical shift imaging sequence after a bolus injection of a hyperpolarized [1-(13)C]pyruvate. Apparent rate constants for the conversion of pyruvate to lactate in three different regions (glioma, normal appearing brain, and vasculature) were estimated based on a two-site exchange model. The apparent conversion rate constant was 0.018 ± 0.004 s(-1) (mean ± standard deviation, n = 6) for glioma, 0.009 ± 0.003 s(-1) for normal brain, and 0.005 ± 0.001 s(-1) for vasculature, whereas the lactate-to-pyruvate ratio, the metabolic marker used to date to identify tumor regions, was 0.36 ± 0.07 (mean ± SD), 0.24 ± 0.07, and 0.12 ± 0.02 for glioma, normal brain, and vasculature, respectively. The data suggest that the apparent conversion rate better differentiate glioma from normal brain (P = 0.001, n = 6) than the lactate-to-pyruvate ratio (P = 0.02).
View details for DOI 10.1002/mrm.24181
View details for Web of Science ID 000311398600022
View details for PubMedID 22334279
In addition to cancer imaging, (13) C-MRS of hyperpolarized pyruvate has also demonstrated utility for the investigation of cardiac metabolism and ischemic heart disease. Although no adverse effects have yet been reported for doses commonly used in vivo, high substrate concentrations have lead to supraphysiological pyruvate levels that can affect the underlying metabolism and should be considered when interpreting results. With lactate serving as an important energy source for the heart and physiological lactate levels one to two orders of magnitude higher than for pyruvate, hyperpolarized lactate could potentially be used as an alternative to pyruvate for probing cardiac metabolism. In this study, hyperpolarized [1-(13) C]lactate was used to acquire time-resolved spectra from the healthy rat heart in vivo and to measure dichloroacetate (DCA)-modulated changes in flux through pyruvate dehydrogenase (PDH). Both primary oxidation of lactate to pyruvate and subsequent conversion of pyruvate to alanine and bicarbonate could reliably be detected. Since DCA stimulates the activity of PDH through inhibition of PDH kinase, a more than 2.5-fold increase in bicarbonate-to-substrate ratio was found after administration of DCA, similar to the effect when using [1-(13) C]pyruvate as the substrate.
View details for DOI 10.1002/nbm.2778
View details for Web of Science ID 000308710400003
View details for PubMedID 22278751